JPS6152286A - Vector and transformant - Google Patents

Abstract

PURPOSE:To obtain a shuttle vector having a high expression efficiency of an exogenote, by inserting the exogenote into a long-chain fragment obtained by cleaving DNA containing a gene of bovine papilloma virus at a specific site. CONSTITUTION:A shuttle vector obtained by inserting an exogenote, preferably human inteferon-gamma into a long-chain fragment obtained by cleaving a DNA vector containing a gene of bovine papilloma virus type I at a PvuII cleavage site. An animal cell is then transformed therewith.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to vectors and transformed cells that secrete expression products of foreign genes in animal cell gene manipulation systems.

More specifically, the present invention relates to so-called shuttle vectors that can be propagated in both animal cells and microbial cells.

[Conventional technology]

In recent years, bovine papillomavirus type I (hereinafter referred to as
Attempts to introduce DNA vectors containing the gene of BPV-1 (abbreviated as BPV-1) into animal cell lines have been widely investigated. (For example l1ukaryotic Viral Vector
s.

Pages 79-9, 19F12 Y, Gluzma
Co1d SpringHarbo
r Laboratory; M, 1. ow, N, 5
arver, P., Ilowly.

Bovine papillomavirus, silk-transformed DN in medical research
A Experiment ■, pp. 69-88, edited by the Medical Research Foundation, 1983). The characteristics of RPV-1 are that when it infects animal cells, it is not integrated into nuclear chromosomes but exists as an extranuclear chromosome, and the number of copies is 10 to 200 copies/cell. 31% were deleted. The so-called B P V69 (Ram III and Hin
It has been reported that a short vector (31% removed by digestion with dm) can infect BPV-susceptible cells derived from the cervix and cause them to become cancerous.

As vectors for foreign gene expression, it is common to use vectors as small as possible that have the minimum functions necessary for replication and maintenance.
``3PVl,,l is used for foreign gene expression.In addition, cells that become cancerous as a result of BPV infection have an accelerated cell division rate, and require expensive serum when subculturing them. It has been reported that it acquires beneficial properties such as reducing

Furthermore, many of the proteins produced are secreted outside the cell.

Based on the properties mentioned above, a genetically engineered substance production system using BPV-infected animal cells as a host and BPV as a vector is expected to have various advantages. In particular, genetically engineered systems using animal cells as hosts are considered to be important for use as production systems for glycoproteins and the like. When complex proteins such as glycoproteins are biosynthesized, DNA-=
The sequence is mRNA → protein - addition of sugars, lipids, etc. - extracellular secretion, but prokaryotes such as Escherichia coli and Bacillus subtilis lack the activity of adding sugars and lipids to the protein. Therefore, even if the glycoprotein gene is expressed using these microorganisms as a host, only the protein portion of the protein will be synthesized.
It has a major drawback that its chemical structure is different. In addition, when E. coli is used as a host, it is necessary to extract it from the bacterial body after two-way culture because the primary substance accumulates within the bacterial body without being secreted. As a result, it has undesirable drawbacks such as a decrease in extraction yield and a decrease in the purity of the target product.

Originally, for the purpose of producing substances that are produced in animal cells, gene manipulation technology that uses animal cells as hosts is a more preferable system, and therefore there is a strong desire to increase the production efficiency of animal cell production systems. This is because it is.

Animal cell expression systems using the BPV-1 gene as a vector have been actively investigated recently, and several cases have been reported in which foreign genes derived from higher animals were expressed using this system. Ma
niatis reported the expression of the human HLA gene.
o1. Ce1. l1io! , 4 (2) :310(
]+984) human IFNβ (Ilowley et al., l'
lo1. cel, old o1.3, 233 (+983))H
B s antigen (Ilofschnelde et al., Mo1.C
e1. Riol, 3.1032 (1983) and the like are known to be expressed.

1 - The above example uses RPV6Q and its llam I
Expression vectors have been produced by utilizing the I1 or 0 l1ind m breakpoint and ligating a foreign gene Y- to that site. However, the current situation is that the above-mentioned vectors do not produce the target protein with sufficient efficiency.

[Problem that the invention seeks to solve]

An object of the present invention is to provide a novel vector that uses BPV-1 hector and has a high efficiency in expressing foreign genes.

[Means to solve the problem]

The present invention relates to bovine papillomavirus type T (BpV).
A long chain fragment (containing 97% of the total BPV-I DNA) obtained by cutting the vector containing gene 1') NA of -1) at the restriction enzyme PvuTI cleavage site is
The object of the present invention is to provide a novel vector with high expression efficiency, which is obtained by inserting a 1llll Kaneko binding.

A large fragment (containing 69% of BPV-1) obtained by digesting BPV-IT)NA with the restriction enzyme Ba1l (I) and the restriction enzymes BamHI and former ndTII (containing +100 of BPV-) ) and a fragment of the E. coli vector pBR322 (containing the origin of replication in E. coli and the ampicillin resistance gene) to create so-called "shuttle vectors" pdBPV-1 and pBPV that can be maintained and propagated in both animal cells and E. coli cells.
697 etc. were constructed by Dr. Ilowley (Pr.
oc, Natl, Acad, Sci,, 79.7147
(1982)), and the vectors used in the present invention are also the above-mentioned two types of vectors provided and provided by Dr. Llowley.

In order to link a foreign gene to the above vector and express it in animal cells as a host, the foreign gene must be linked downstream of a promoter that transcribes it into mRNA. The promoter is preferably an animal virus gene promoter or an animal cell gene promoter. - Examples include SV40 early promoter, SV40 late promoter, adenovirus gene promoter, HB virus gene promoter, retrovirus gene promoter, etc. as animal virus gene promoters. Further, promoters of animal cell genes include thymidine kinase gene promoters, metallothionein gene promoters, interferon gene promoters, and the like. In the examples of the present invention, an example using the 5v40 early promoter will be described, but the present invention is not limited thereto.

The protein or peptide encoded by the foreign gene used in the present invention is produced in animal cells by using the hector of the present invention. The protein or peptide is not particularly limited, and usually includes useful physiologically active proteins, enzymes, peptides, and the like. In the present invention, Inter 21
0710 example is shown, but of course the present invention is not limited thereto.

There are many methods for linking and inserting a foreign gene with 9 expression function into a BPV-1 vector. Ram BPV-1
After removing 31% of the DNA fragment by digestion with HT and ll1ndlH, the remaining 69% fragment retains Hector's basic property of sustaining and proliferating in cells, so foreign genes are often inserted into this fragment to inhibit expression. It was done.

As an expression vector.

It is common practice to use the smallest possible hector that has the minimum functionality necessary for replication and maintenance, as this has the advantage of ease of use. Therefore, even after inserting a foreign gene, 1
The usual method is to make it as long as the original hector. However, in the present invention, P, M, Ilow
Using the pdBPV-1 hector distributed by Ley, we introduced a foreign gene into the middle of the BPV-derived DNA, and investigated a method for creating a hector that leaves the BPV gene intact as much as possible. It was discovered that Hector can be obtained with much higher expression efficiency than conventional Hector.

The preferred position for inserting the foreign gene is between the namT (T) and ll1ndTII cleavage sites, where the hector function will not be lost even if removed. There are two restriction enzyme PvuTI cleavage sites close to each other near the rlamHr of BPV-1. It exists (Figure 1).

Since there is no other Pvun cleavage site in pdBPV-1, linear DNA is generated by PvuII digestion.
was obtained, which was a preferred hector fragment (10.3 kb fragment) containing 97% of the BPV-1 gene. As understood from FIG. 1, this fragment is a DNA fragment derived from BPV-1 with only the peripheral region deleted (92-95%) and is a DNA fragment with a low degree of modification.

By ligating the foreign gene linked to the promoter described above to the DNA fragment thus obtained, the production of a high expression hector using a BPV vector is completed based on the present invention. The method of binding the promoter to the target protein and the method of inserting it into the fragment described in 1-1 are as follows:
By using the target gene, base sequence, restriction enzyme map, and if necessary, synthetic nucleotides, etc., it can be easily designed and implemented by those skilled in general gene manipulation techniques. An example in which the IFNr gene is linked to the SV40 early promoter will be described below, but of course the present invention is not limited thereto.

Plasmid pIFNγ15, in which the IFNr cDNA was cloned into pBR322, was digested with BalIIH■ to obtain a 6 kb DNA fragment, which was digested with Dpnl to obtain 8006 DNA containing the IFNr gene. The 5V-40 promoter is the psV2rabbit βglobin produced by Mulligan et al. with 1lindTIT and 8g7! A fragment containing the SV40 promoter, SV40 replication origin, pBR322 replication origin, and ampicillin resistance gene was obtained by digestion with n.

Both fragments thus obtained were ligated using T4 ligase, E. coli was recombined using the obtained plasmid, and screening was performed using a restriction enzyme cleavage map of the plasmid to obtain the target plasmid psVIFNγ, that is, SV
A target plasmid having IFNr downstream of the 40 promoter was obtained. When psVTFNr was digested with Accn, a fragment of the IFNγ gene containing the desired SV40 promoter was obtained. T)N containing 97% of the previously obtained BPV-1
IFN containing the A fragment and the SV40 promoter obtained here
By ligating the γ gene fragment with T4 ligase, the foreign gene IFNγ is inserted into the Pvu11 cleavage site of the live papillomavirus type I gene DNA related to the present invention.
(containing the SV40 promoter)
FNr expression vector psVIFNγ/BPV9? was successfully created.

The expression vector obtained here is pB in E. coli.
It is a so-called Shall vector, which is maintained in replication by a portion derived from R322, and in animal cells by a portion derived from BPV. Therefore, in order to obtain a large amount of this vector, it is preferable to culture, propagate, isolate and purify it using E. coli as a host. Expression of the inserted gene into protein is impossible in E. coli because an animal cell promoter is used as the promoter, and eukaryotic cells must be used as the host.

By recombining eukaryotic cells using this vector obtained in large quantities in Escherichia coli, recombinant cells that produce the target protein can be obtained. Mouse-derived C-127 cells, NIH3T3, and the like are known as eukaryotic cells in which a plasmid containing BPV-I DNA can be stably maintained and propagated.

Conventional methods are sufficient for introducing vector DNA into animal cells, such as the standard calcium phosphate method.

Cells transformed with a vector containing BPV-I DNA became cancerous, and the rate of cell division was markedly increased.

As a result, only the recombinant cells form a so-called focus with a high cell density on the soft agar nutrient medium, and this property has the advantage that it is easy to sort silk-modified cells from the original host cells. There is.

In order to select recombinant cells, a vector containing a drug resistance gene and a vector containing BPV-I DNA are mixed, Co-Transformation L is performed, and recombinant cells that have become resistant to the drug are screened.

Recombinant cells can be selected by utilizing the fact that the recombinant cells are also frequently transformed with BPV vectors. Cells that can be transformed with a vector containing BPV-I DNA include mouse-derived C127 cells or NIH
Cells in which a vector containing BPV-1 can be stably maintained, such as 3T3 cells, are preferably used.

Cells transformed with the BPV-1 vector according to the invention grow in a normal medium, such as Dulbecco's MEM medium, and secrete the protein of interest, such as TFNr, into the medium. The recombinant cells retained the advantageous properties observed in the BPV-]669% vector, such as rapid division and markedly reduced serum requirement.

[Results of the invention]

The vector of the present invention is a BPV-1 vector with high expression efficiency. For the expression of human interferon γ shown in this example, human tonsil-derived lymphocytes were mixed with TPA and PHA.
The expression was extremely high, about 30 times the amount synthesized when treated with -M.

The present invention will be specifically described below with reference to Examples.

Example 1 Preparation of plasmid psVIFNT having the IFNr gene downstream of the SV40 promoter Plasmid pTFNr-15 in which the IFN'r gene was cloned into pBR322 (Japanese Patent Application No. 59-13)
A 1.6 kb DNA fragment containing the TFNγ gene obtained by digesting the DNA fragment (No. 2 9398) with Bam old was digested with Dpnl and subjected to agarose gel electrophoresis. O, Rkb I
A band of DNA fragments containing the FNγ gene was cut out, and nine pieces of DNA were eluted from the gel by electroelution, agarose was removed by Millipore filter filtration, phenol-chloroform treatment, and ether treatment, and purified by ethanol precipitation.

Next, p S V 2 rahbitβglobin(R
,C,Mulliganet al,5science
Kario 1422 (1980)) is the former nd m, Rg
Of the two DNA fragments obtained by digestion with R■.

Longer DNA fragment (SV, 10pt) motor, 5V
40ori, pBR322ori, Am resistant, etc.) were purified by agarose gel electrophoresis and electroelution method as described above. This DNA fragment was dephosphorylated by bacterial alkaline phosphatase (BAP) treatment, BAP was inactivated by phenol-chloroform treatment, and purified by ethanol precipitation.
, DNA polymerase T1. a
After making the ends blunt with rge Prag-ment, 0.8k containing this and the previously obtained IFNγ gene
The DNA fragment of b was ligated with T4 ligase. Escherichia coli MC1061 (J, Mo1. Riot, 138
．． 179 (1980)).

Transformants were obtained at a frequency of 5 x 103 cells/ng DNA.

Twelve transformants were randomly selected from the obtained transformants, plasmid DNA was prepared by alkaline SDS method, and the desired plasmid p was extracted by restriction enzyme treatment and agarose gel electrophoresis.
A search was made for strains containing SVI FNT.

As a result, one strain retained the desired plasmid.

Example 2 Vector pSV TF N ! in which the TFNγ gene was linked to the Pvu cleavage site of BPV-1. / B P
V9? Construction of pBPV-1 as a vector containing the BPV-1 gene
(142-6> was used. (P, M,) Iowley
et, al, +Proc, Na t I, ^ca
d, Sci., 79, 7] 47 (1982)).

The procedure for producing psVrFNγ/BP V Q 7 is shown in FIG.

i) Preparation of DNA from pdB PV 1 (142-6): pdB PV -1 (+42-6)
was digested with Pvull. 1 by agarose gel electrophoresis
An agarose gel containing a 0.3 kb DNA fragment was cut out. DNA fragments were purified by electroelution in the same manner as in Example 1. The obtained DNA fragment was dephosphorylated with BAP.

After BAP was inactivated by phenol-chloroform treatment, it was purified by ether treatment and ethanol precipitation.

ii) Preparation of DNA fragment from pdSV TFNr: psVIFNr was digested with AccH and an agarose gel containing a 2.9 kb DNA fragment was excised by agarose gel electrophoresis. Same as Example 1.

DNA fragments were purified by electroelution.

1ii) psVrFNr/BPVq7(7) construction:
0.3μg of the 10.3kb DNA fragment prepared in i) and 0.1iig of the 2.9kb DNA fragment prepared in ii)
T4T) NA ligase 5f(() at 10 °C
The mixture was allowed to react overnight. M Cl0 using the z-view of the reaction solution
61 competent cells were transformed according to the standard method, and 1.4
Colonies were obtained at a frequency of xlO'co/μg DNA. From the obtained colonies, 24 colonies were picked up, plasmid DNA was prepared by the alkaline SDS method, and the target plasmid f) SVI was extracted by agarose gel electrophoresis.
FNr/BPVqt (71 strains were searched.

As a result, 3 out of 24 strains retained the target plasmid.

Example 3 Transformation of mouse Cl27 cells with pSVTFNr/BPVq7 Human IFNr expression vector psV prepared in Example 2
Vector pSV2-ne containing 70.5 μmol of TFNT/BPVq and a neomycin resistance gene.

(D, J, 5outhern et al, JoM
ol, 8pp1. Genet, 1+3
27.1982) O, Ipmo+ using the calcium phosphate method (1', L.

Graham et al, Virology 54.
536.1973), approximately 5×10 0127 cells (D, R, 1, owy et al, J, Vi-ro
191.1978). 1600
/7 g/ml G41B (GTBCO) and Darhesoko MEM medium (Hinaga Pharmaceutical@) containing 101% tallow serum was cultured for 17 days, resulting in 26 0418-resistant transformations. I got a body. Of these, 17 clones were separated into a 96-well microplate (Corning). Culture the cells until they become confluent,
When the interferon activity in the medium was measured after 2 to 4 days, a recombinant cell line was obtained that produced human interferon γ at a maximum level of 20,300 levels/1 or more. The results are shown in Table 1.

In addition, the quantification of interferon activity is performed using S, Ru-b
The method of instein et al. (J, Virology, 3
7.755.1981), human FL cells, 5
It was carried out using the CPE50 method using indbis and viruses. As the standard interferon, recombinant Escherichia coli-derived human interferon γ8300tF, -position/1, which had been subjected to NIH standard human interferon typing, was used.

Table 1 Example 4 Ability of Transformants to Produce Human Interferon γ in Serum-Containing Medium Among the transformants obtained in Example 3, clones with high human interferon T production ability were added to the medium every 2 to 4 days. The interferon activity secreted into the medium was measured over 42 days while replacing the medium.

All clones remained viable and continued to produce interferon with only medium changes.

The most active clone, 11h64-1, produced 1.8 million units of human interferon γ per approximately 10 6 cells for a total of 42 days. The results are shown in Figure 2.

Example 5 Human interferon γ production performance of transformants in serum-free medium Clone It164-1 obtained in Example 4 was cultured in Dulbecco's MEM medium supplemented with 10% tallow serum and Dulbecco's MEM medium without side edges. After culturing for 9 days, the amount of interferon produced and the number of cells reached were examined.

The number of cells reached, that is, the cell proliferation rate, was reduced to 80% of that in serum-containing medium when using serum-free medium, but interferon production per cell remained unchanged. Table 2 shows the results.

Example 6 Physical properties of human interferon γ produced by transformants 90 units of interferon produced by clone Na64-1 obtained in Example 4 were treated with 3nl of anti-human interferon T antibody (neutralizes human interferon γ at position 84) ) or anti-human interferon β antibody 12.5n
When the residual interferon activity was measured after treatment with 1 (neutralizes human interferon β at position 56044) at 37°C for 30 minutes, it was found to be specifically neutralized by anti-human interferon T antibody.

Furthermore, when interferon at position 66 was treated under p112 conditions at 4°C for 30 minutes, residual interferon activity was measured and was below the detection limit (18 units).

In this way, it is neutralized by antibodies. The property of being inactivated by acid is a property specific to human interferon γ. The measurement results are shown in Table 3.

Reference Example-1 Human TFNγ production ability of C127 cells transformed with pSVIFNγ/BPV 69T BPV9? used in Example 2? instead of B P V69
Human IFNγ-expressing Hector pSVI prepared using T.
F N r / B P V 69 T O, 5 pmol
and psV2-ne.

O, 1 pmol and about 5×1 by calcium phosphate method.
05 0127 cells. 1600 #g/
When cultured for 15 days in Dalhecco's MEM medium containing G41B and 10% tallow serum, 14
A transformant resistant to 418 was obtained. 9 of these
Separate into a 6-well microplate and make sure the cells are confluent.
The cells were cultured for 4 to 7 days until reaching nt. The TFN activity in the thus obtained medium was measured for 9 clones and was found to be 2000 to 4001 positions/ml. This is the I of clones 32-1 and 64-1 shown in Example 3.
The amount of FN produced is 18 or less.

The measurement results are shown in Table 4.

Table 4

[Brief explanation of drawings]

FIG. 1 shows the Hector psVIFNγ/BPV9? of the present invention.
FIG. 2 is a graph showing the integral value of the human IFNr titer secreted into the medium when clone No. 64-1 of Example 4 was maintained only by medium exchange.

Claims (3)

[Claims] (1) Cutting a DNA vector containing the bovine papillomavirus type I gene at the PvuII cleavage site,
A vector made by inserting a foreign gene into the obtained long chain fragment. (2) The vector according to claim (1), wherein the foreign gene is human interferon-γ. (3) Cutting a DNA vector containing the bovine papillomavirus type I gene at the PvuII cleavage site,
A transformant obtained by transforming animal cells with a vector obtained by inserting a foreign gene into the obtained long-chain fragment.